The response to heat stress is ancient and highly conserved. In the animals it frequently results in apoptosis, or if the initial stress is sub-lethal, resistance to subsequent heat shock. While readily demonstrated, the mechanisms of heat shock-induced apoptosis and its regulation by heat shock proteins are not well understood. Based on an emerging paradigm of initiator caspase activation and a novel method to detect such activation, we have found that heat shock-induced apoptosis is initiated by a novel pathway involving caspase-2. In contrast, a variety of other stressors, including those working by death receptor ligation, DNA damage, endoplasmic reticulum stress, and others, fail to engage caspase-2, and instead initiate apoptosis via either caspase-8 or caspase-9 activation. Our results are supported by preliminary results from caspase-2-deficient mice. The aims we will address follow from these considerations. These are: 1. To investigate the activation of caspase-2 following heat shock and its role in heat shock-induced apoptosis. We will investigate the mechanisms involved in the activation of caspase-2 by heat shock and related stress, and how this leads to the death of the cell. Our studies will employ new approaches to the detection of initiator caspase activation, based on emerging concepts of caspase function. Proteins known to interact with caspase-2 will be examined for their possible roles in heat shock-induced apoptosis, as will proteins we find in association with caspase-2 during this process. 2. To determine how thermotolerance and heat shock-proteins function to block heat shock-induced apoptosis. Cells that are subjected to sub-lethal heat stress become resistant to subsequent heat shock, through the expression of heat shock proteins, especially HSP70. We will examine this resistance with respect to apoptosis and the activation of caspase-2, and compare it with other mechanisms such as expression of the anti-apoptotic Bcl-2 family members. Effects of removing or down-regulating HSPs will be examined in terms of potential sensitization to caspase-2 activation. 3. To explore how heat shock and caspase-2 activation engage the mitochondrial pathway of apoptosis. Caspase-2 does not directly process executioner caspases or their substrates, and therefore how heat shock and caspase-2 activation kill cells is not obvious. We will probe the role of pro-apoptotic Bcl-2 family members and the mitochondrial pathway in this process. In particular, we will investigate how caspase-2-mediated cleavage of Bid may synergize with direct effects of heat stress on other Bcl-2 family members (Bax and Bak) to promote apoptosis via the mitochondrial pathway. These three goals represent independent and complimentary approaches to the elucidation of the novel apoptotic pathway represented by heat shock and proceeding through the activation of caspase-2 [unreadable] [unreadable] [unreadable]